A doença meningocócica na região de Sorocaba, São Paulo, Brasil, no período de 1999 a 2008

O objetivo do estudo foi descrever a ocorrência da doença meningocócica notificada no Departamento Regional de Saúde XVI, Sorocaba, São Paulo, Brasil, no período de 1999 a 2008. Em Sorocaba, a incidência anual da doença foi de cerca de dois casos por 100 mil habitantes, com aumento entre 2006 e 2008. As incidências anuais foram maiores entre 0 a 4 anos de idade. A letalidade no período foi de 21,8%, maior nas faixas etárias de 0 a 4 anos (26,4%), na qual se deu a maior incidência da doença, e com idade superior a 30 anos (28%). A confirmação diagnóstica foi laboratorial em 71% dos casos (cultura em 45,3%) e por critérios clínico-epidemiológicos em 22%. O sorogrupo B ocorreu em 45,7%; o C, em 47,3%; o W135, em 3,7%; e o Y, em 1,5% dos casos identificados, com predomínio do sorogrupo B, entre 1999 e 2003, e do C, entre 2004 e 2008. Os fenótipos B:4,7:P1.19,15 e C:23:P1.14-6 predominaram. Os resultados reforçam a necessidade de acompanhamento, de forma regional, da tendência da doença para a detecção de surtos precocemente e monitoramento de cepas circulantes.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

Reticulate eruptions. Part 1: Vascular networks and physiology

Reticulate pattern is one of the most important dermatological signs of a pathological process involving the superficial vascular networks. Vascular malformations, such as cutis marmorata congenita telangiectasia and benign forms of livedo reticularis, and sinister conditions, such as meningococcal meningitis or Sneddon's syndrome, can all present with a reticulate pattern. The clinical presentation and morphology is determined by the nature and extent of the underlying pathology and the involvement of a particular vascular network. This review has been divided into four instalments. In the present paper, we discuss the anatomy and physiology of the complex network of vascular structures that support the function of the skin and subcutis.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Eosinophils in the gastrointestinal tract: friends or foes?

Eosinophils play an important role in the mucosal immune system of the gastrointestinal tract under resting and under inflammatory conditions. Under steady-state conditions, the mucosa of the digestive tract is the only organ harboring a substantial number of eosinophils, which, if need be, get activated and exert several effector and immunoregulatory functions. The precise function of these late-phase inflammatory cells is not yet completely understood. Nevertheless, it has recently been demonstrated that lipopolysaccharides from gram-negative bacteria activate eosinophils to rapidly release mitochondrial DNA in the extracellular space. Released mitochondrial DNA and eosinophil granule proteins form extracellular structures able to bind and inactivate bacteria. These findings suggest a novel mechanism of eosinophil-mediated innate immune responses that might be important in maintaining the intestinal barrier function. Moreover, eosinophils also play a crucial role in several inflammatory conditions, such as intestinal infections, immune-mediated inflammations and hypersensitivity reactions. Under chronic inflammatory conditions, the ability of the eosinophils to induce repair can lead to pathological sequelae in the tissue, such as esophageal remodeling in eosinophilic esophagitis. It is established that the uncontrolled eosinophilic inflammation induces fibrosis, esophageal wall thickening and strictures leading to damage that results in a loss of esophageal function. One potential mechanism of this remodeling is so-called 'epithelial mesenchymal transition', which is triggered by eosinophils and is potentially reversible under successful anti-eosinophil treatment. Therefore, eosinophils may act either as friends or as foes, depending on the microenvironment.

Strategies to prevent neuronal damage in paediatric bacterial meningitis

PURPOSE OF REVIEW: The mortality of bacterial meningitis can reach 30%, and up to 50% of survivors suffer from persisting neurological deficits as a consequence of the disease. The incidence of neurological sequelae of bacterial meningitis has not improved over the last decade. Adjunctive therapeutic options are limited, and ongoing research into the pathophysiology of brain damage in bacterial meningitis aims at providing the scientific basis for future development of more efficient adjunctive options. RECENT FINDINGS: In a population with good access to health care, dexamethasone given before or at the time of initiation of antibiotic therapy acts beneficially in paediatric pneumococcal meningitis, but not in meningococcal meningitis. In experimental animal models, brain-derived neurotrophic factor protected against brain injury and improved hearing while melatonin, which has antioxidant properties among other effects, reduced neuronal death. Transgene technology can be used to provide new insights into the pathophysiology of the disease and to identify potential therapeutic targets. SUMMARY: Although dexamethasone improves outcome of bacterial meningitis under defined circumstances, the morbidity of bacterial meningitis still remains unacceptably high. Experimental models may help to identify new therapeutic strategies to further improve the neurological outcome in young children suffering from bacterial meningitis.

[Acute infectious emergencies in adults in medical practice]

Infectious diseases belong to the most frequent reasons to seek emergency care. Life-threatening infectious emergencies, which require rapid diagnosis and hospitalisation, are, however, rare. Leading signs and symptoms are high fever combined with rapidly deteriorating general conditions, hypotonia, tachycardia, tachypnea, dyspnea, confusion, headache, or petechia or information about asplenia, immunosuppression or recent travel to the tropics. Life threatening situations, such as suspicion of invasive meningococcal infection or bacterial infection in an asplenic patient, septic-toxic shock, and acute bacterial meningitis with delayed hospitalisation require rapid start of empiric antibiotic therapy in the outpatient practice. In addition, acute infectious emergencies comprise situation for which post exposure prophylaxis is indicated.

Effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) on cerebrospinal fluid inflammation induced by endotoxin

Endotoxin triggers the subarachnoid inflammation of gram-negative meningitis. This study examined the ability of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) to block endotoxin-induced meningitis in rabbits. Intracisternal (ic) injection of 10-20 ng of meningococcal endotoxin induced high cerebrospinal fluid (CSF) concentrations of tumor necrosis factor (TNF) and CSF pleocytosis and increased CSF lactate concentrations. ic administration of rBPI23 significantly reduced meningococcal endotoxin-induced TNF release into CSF (P < .005), lactate concentrations (P < .001), and CSF white blood cell counts (P < .01). No such effect was observed in animals receiving intravenous rBPI23. Concentrations of rBPI23 in CSF were high after ic administration but low or undetectable after systemic administration. Thus, high concentrations of rBPI23 can effectively neutralize meningococcal endotoxin in CSF, but low CSF concentrations after systemic administration currently limit its potential usefulness as adjunctive drug treatment in gram-negative meningitis.

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

A direct role for secretory phospholipase A2 and lysophosphatidylcholine in the mediation of LPS-induced gastric injury.

Endotoxemia from sepsis can injure the gastrointestinal tract through mechanisms that have not been fully elucidated. We have shown that LPS induces an increase in gastric permeability in parallel with the luminal appearance of secretory phospholipase A2 (sPLA2) and its product, lysophosphatidylcholine (lyso-PC). We proposed that sPLA2 acted on the gastric hydrophobic barrier, composed primarily of phosphatidylcholine (PC), to degrade it and produce lyso-PC, an agent that is damaging to the mucosa. In the present study, we have tested whether lyso-PC and/or sPLA2 have direct damaging effects on the hydrophobic barriers of synthetic and mucosal surfaces. Rats were administered LPS (5 mg/kg, i.p.), and gastric contents were collected 5 h later for analysis of sPLA2 and lyso-PC content. Using these measured concentrations, direct effects of sPLA2 and lyso-PC were determined on (a) surface hydrophobicity as detected with an artificial PC surface and with intact gastric mucosa (contact angle analysis) and (b) cell membrane disruption of gastric epithelial cells (AGS). Both lyso-PC and sPLA2 increased significantly in the collected gastric juice of LPS-treated rats. Using similar concentrations to the levels in gastric juice, the contact angle of PC-coated slides declined after incubation with either pancreatic sPLA2 or lyso-PC. Similarly, gastric contact angles seen in control rats were significantly decreased in sPLA2 and lyso-PC-treated rats. In addition, we observed dose-dependent injurious effects of both lyso-PC and sPLA2 in gastric AGS cells. An LPS-induced increase in sPLA2 activity in the gastric lumen and its product, lyso-PC, are capable of directly disrupting the gastric hydrophobic layer and may contribute to gastric barrier disruption and subsequent inflammation.

Ketamine-induced hepatoprotection: the role of heme oxygenase-1.

Lipopolysaccharide (LPS) causes hepatic injury that is mediated, in part, by upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Ketamine has been shown to prevent these effects. Because upregulation of heme oxygenase-1 (HO-1) has hepatoprotective effects, as does carbon monoxide (CO), an end product of the HO-1 catalytic reaction, we examined the effects of HO-1 inhibition on ketamine-induced hepatoprotection and assessed whether CO could attenuate LPS-induced hepatic injury. One group of rats received ketamine (70 mg/kg ip) or saline concurrently with either the HO-1 inhibitor tin protoporphyrin IX (50 micromol/kg ip) or saline. Another group of rats received inhalational CO (250 ppm over 1 h) or room air. All rats were given LPS (20 mg/kg ip) or saline 1 h later and euthanized 5 h after LPS or saline. Liver was collected for iNOS, COX-2, and HO-1 (Western blot), NF-kappaB and PPAR-gamma analysis (EMSA), and iNOS and COX-2 mRNA analysis (RT-PCR). Serum was collected to measure alanine aminotransferase as an index of hepatocellular injury. HO-1 inhibition attenuated ketamine-induced hepatoprotection and downregulation of iNOS and COX-2 protein. CO prevented LPS-induced hepatic injury and upregulation of iNOS and COX-2 proteins. Although CO abolished the ability of LPS to diminish PPAR-gamma activity, it enhanced NF-kappaB activity. These data suggest that the hepatoprotective effects of ketamine are mediated primarily by HO-1 and its end product CO.